This invention relates to an enzymatic reagent, kit and method for determining the concentration of monovalent anions, especially chloride, particularly in biological fluids, and especially in a single reagent format.
The determination of chloride is routinely made in a wide variety of fluids, e.g., industrial solutions, swimming pools, and in clinical chemistry. The last mentioned is of particular importance because chloride is the major extracellular anion and is therefore significantly involved in the maintenance of water distribution, extracellular electrolyte balance and osmotic pressure. Hypochloremia is observed in Addisonian crisis, certain metabolic acidoses, prolonged vomiting and in metabolic alkalosis. Hyperchloremia is common in dehydration, renal tubular acidosis, acute renal failure, prolonged diarrhea and salicylate intoxication (Tietz, N. W., Textbook of Clinical Chemistry, p. 1183, W. B. Saunders Co., Philadelphia, PA, 1986).
Clinically, chloride is commonly measured in urine, serum or plasma, spinal fluid and sometimes in sweat. The most common methods for chloride analysis are mercurimetric titration (Schales, O., and Schales, S. S., J. Biol. Chem., 140, 879-884, 1941.), coulometric titration (Cotlove, E., In: Methods of Biochemical Analysis, Vol. 12. D. Glick, Ed. New York, Interscience Publishers, Inc., 1964.), spectrophotometry (Zall, D. M., Fisher, D., and Garner, M. Q., Anal. Chem. 28, 1665-1668, 1956; West, P. W., and Coll, H., Anal. Chem. 28, 1834-1838, 1956; Schoenfeld, R. G., and Lewellen, C. J., Clin. Chem. 10, 533-539, 1964; Hamilton, R. H., Clin. Chem. 12, 1-17, 1966; Law, W. T., and Ertingshausen, G., Clin. Chem. 26, 1874-1877, 1980 [U.S. Pat. No. 4,278,440].), ion selective electrode methodology (Dahms, H., Rock, R., and Seligson, D., Clin. Chem. 14, 859-870, 1968; Rockenmacher, M., Am. J. Clin. Path. 33, 349-354, 1960.) and a recently developed enzymatic assay (Ono, T., Taniguchi, J., Mitsumaki, H., Takahata,.F-, Shibuya, A., Kasahara, Y., and Koshimizu, F., Clin. Chem. 34, 552-553, 1988 [U.S. Patent Pending].), but all of these assays suffer from at least one important disadvantage.
Coulometric methods are widely used but time consuming maintenance procedures have reduced their appeal. Ion selective electrodes are becoming the method of choice in most laboratories but they too require extensive maintenance. The mercurimetric titration and most of the spectrophotometric methods contain mercury which creates waste disposal and environmental problems. All of the spectrophotometric methods contain strong acids which necessitate handling precautions and make them unsuitable for tabletting.
The newly developed amylase-based enzymatic assay, as described, is not suitable for unit dose analyzers due to the requirement for a considerable incubation period before the addition of a second reagent for initiating the reaction.